hNOA1 is a large GTP-binding protein. A, domain organization of human hNOA1. MTS, mitochondrial targeting sequence; CC, coiled coil; LZ, leucine zipper; GTPase, GTPase domain. B, immunoblot showing the expression of hNOA1 in rat tissues. hNOA1 is ubiquitously expressed in all tissues tested with slightly higher expression in heart (H), kidney (K), and lung (Lu) followed by skeletal muscle (SM) and liver (Li), and the lowest expression in pancreas (P) and spleen (Sp). In brain (B), a slightly higher band was detected. Equal aliquots (∼120 μg of protein) of each tissue lysate were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with anti-hNOA1 IgG. C: Upper panel, hNOA1 binds [α-³²P]GTP in vitro.[α-³²P]GTP binds to His₆-Gα_i3 (lane 1) and His₆-hNOA1 (lane 2), but not BSA (lane 3). The ∼40-kDa band in lane 2 is most likely a degradation product of His₆-hNOA1. Equal amounts (1.5 μg) of His₆-Gα_i3, His₆-hNOA1, and BSA shown by Coomassie Blue staining (lower panel) were incubated with [α-³²P]GTP (upper panel), followed by UV cross-linking, SDS-PAGE, and autoradiography.

Endogenous hNOA1 is localized on mitochondrial cristae. A-C, immunofluorescence showing endogenous hNOA1 colocalizes with the mitochondrial marker cytochrome c (Cyt C) in COS7 cells (yellow in merge image). D-F, endogenous hNOA1 does not colocalize with the Golgi marker ERGIC-53. COS7 cells were fixed in 2% PFA and incubated with affinity-purified rabbit anti-hNOA1 IgG (1:500) and mouse anti-cytochrome c (1:1000) or anti-ERGIC-53 (1:250) IgG, followed by goat anti-rabbit Alexa Fluor-594 (1:500) and anti-mouse Alexa Fluor-488 (1:500) F(ab′)₂, and examined by immunofluorescence. G, immunogold labeling demonstrating that hNOA1 is associated largely with the cristae (arrows) but not the OMM (arrowheads). Ultrathin cryosections of rat kidney were prepared as described under “Experimental Procedures” and labeled with anti-hNOA1 IgG followed by 10 nm gold, goat anti-rabbit IgG conjugates. Bar = 1 μm.

hNOA1 is peripherally associated with the IMM facing the matrix. A, hNOA1 and ATP synthase (mitochondrial marker) are enriched in purified rat liver mitochondria (lane 1), which lack PMP70 (peroxisome marker) and protein disulfide isomerase (PDI) (ER marker). PMP70 and PDI are detected in a crude mitochondrial fraction (lane 2). Mitochondria were prepared from rat liver by differential centrifugation and further enriched by Histodenz gradient centrifugation. ∼20 μg of the crude (lane 2) and purified (lane 1) fractions were separated by 10% SDS-PAGE and immunoblotted for hNOA1, ATP synthase, PMP70, or PDI. B, hNOA1 is not released by mitochondrial swelling. hNOA1, Opa1, and DAP3 are found exclusively in the pellet (P)(lane 3), which contains proteins on or associated with the IMM and matrix. A fraction of the cytochrome c was released into the supernatant (S) with the soluble IMS proteins (lane 2) as expected. ∼150 μg of mitochondria prepared from HEK293 cells by differential centrifugation were swollen with hypotonic medium to break the OMM, keeping the IMM intact. The swollen mitochondria were pelleted by centrifugation, the supernatant (S) containing the soluble proteins released from the IMS was collected, and the pellet (P) was suspended in the same volume as the supernatant. Equal volumes of supernatant and pellet were separated by 12% SDS-PAGE, followed by immunoblotting for hNOA1, cytochrome c, DAP3, and Opa1. C, hNOA1 and DAP3 are resistant to trypsin digestion, whereas Opa1 is susceptible to trypsin digestion of swollen mitochondria (lane 2). Digestion of Opa1 is expected based on its localization on the IMM facing the intermembrane space. After adding 1% Triton X-100, hNOA1, Opa1, and DAP3 are digested by trypsin (lane 3). Mitochondria, prepared and swollen as in B, were incubated with or without 25 μg/ml trypsin and/or 1% Triton X-100 for 20 min. After incubation, the trypsin activity was stopped by addition of soybean trypsin inhibitor, and samples were immunoblotted for hNOA1, DAP3, and Opa1. D, hNOA1 is extracted by sodium carbonate (pH 11.5), but not by high salt (KCl). hNOA1, Opa1, and DAP3 remain in the pellet (P) after the high salt (KCl) wash (lane 2), but are extracted and released into the supernatant (S) after sodium carbonate treatment (pH 11.5, lane 3). As a control, cytochrome c can be extracted by both high salt (KCl) and sodium carbonate (pH 11.5) treatment (compare lanes 4 and 5 with lanes 2 and 3). COX I, an integral membrane protein, remains in the pellet after high salt or sodium carbonate (pH 11.5) treatment (lanes 2 and 3). A swollen mitochondrial pellet prepared as in B, was suspended and washed in either high salt or sodium carbonate (200 mm KCl) as described under “Experimental Procedures.” After extraction, the suspension was centrifuged to yield the supernatant (S) and pellet (P), which was suspended in the same volume as the supernatant. An equal volume of each fraction was immunoblotted for hNOA1, Opa1, DAP3, Cox I, and cytochrome c.

Changes in hNOA expression affect mitochondrial dynamics. A-C, in COS7 cells with high levels of hNOA1 expression (asterisk), mitochondria as marked by cytochrome c (Cyt C) staining often become small and round. D-F, in uninduced cells (triangles) mitochondria are more filamentous (see also Fig. 2, A-C). Tet-on hNOA1-inducible stable COS7 cells were treated or not with 1 μg/ml doxycycline for 4 days, followed by fixation and immunofluorescence as in Fig. 2, A-C. G, in control COS7 cells (-DOX), ∼95% of the cells show normal filamentous mitochondria and <5% have small round mitochondria. After induction of hNOA1 expression (+DOX), ∼41% of the cells show small round mitochondria. 20,000 Tet-on hNOA1-inducible stable COS7 cells were plated per well in 12-well plates with or without 1 μg/ml doxycycline for 4 days. Cells were fixed and processed for immunofluorescence with anti-hNOA1 and anti-cytochrome c IgG. The total number of cells in 12 randomly selected fields (250 for +Dox; 338 for -Dox) were counted based on 4′,6-diamidino-2-phenylindole nuclear staining, and those with small and round mitochondria were counted. H, immunoblot showing that hNOA1 is efficiently induced after addition of 1 μg/ml doxycycline (+Dox) for 4 days. hNOA1 Tet-on-inducible COS7 cells plated in 12-well plates were treated with (+Dox)(lane 2) or without (-Dox)(lane 1) doxycycline for 4 days. After induction, cells were lysed directly on the plate with 2× sample buffer followed by 10% SDS-PAGE and immunoblotting with anti-hNOA1 and anti-actin IgG. I, immunoblot showing that hNOA1 is efficiently depleted by siRNA specific for hNOA1. COS7 cells were transfected with control (lane 1) or hNOA1-specific siRNA (lane 2) using Oligofectamine. After 3 days of transfection, cells were lysed directly in sample buffer, followed by immunoblotting with anti-hNOA1 and anti-actin IgG. J and K, COS7 cells were similarly transfected with control (J) or hNOA1-specific (K) siRNA and examined by electron microscopy. In cells transfected with hNOA1 siRNA, the mitochondria (m) appear larger, and the mitochondrial matrix has a lower density than in controls. Bar = 1 μm.

hNOA1 interacts with Complex I of the electron transport chain and regulates NADH-linked mitochondrial O₂ consumption. A, silver-stained gel of immunoprecipitates obtained with anti-hNOA1 or preimmune (Pre) IgG. Bands present only in the hNOA1 immunoprecipitates (arrows) were identified by mass spectrometry as hNOA1, Complex I, 75 kDa (CI-75 kDa), DAP3/Mrp S29, Mrp S27, and Mrp L12. ∼1 mg of enriched mitochondria prepared from HEK293 cells by differential centrifugation were incubated with 40 μg of anti-hNOA1 or preimmune IgG. Immune complexes were bound to 30 μl of protein A-Sepharose beads and separated by 10% SDS-PAGE, followed by silver staining. B, hNOA1 coimmunoprecipitates with the 39 kDa (CI-39kD) and 30 kDa (CI-30kD) subunits of Complex I, but not subunit IV of Complex IV. Immunoprecipitates obtained with anti-hNOA1 and preimmune (Pre) IgG were separated by SDS-PAGE and immunoblotted for hNOA1, CI-39 kDa, CI-30 kDa and subunit IV of Complex IV. C, GST-hNOA1 interacts with the 39-kDa subunit of Complex I in GST-pulldown (PD) assays. Immobilized recombinant GST or GST-hNOA1 (∼2 μg each) were incubated with HEK293 cell lysates for 4 h, followed by washing with lysis buffer. Upper panel, proteins bound to immobilized fusion proteins were eluted with 2× sample buffer for SDS-PAGE and immunoblotted with anti-CI-39 kDa IgG. Lower panel, Coomassie Blue staining showing the amount of GST or GST-hNOA1 fusion proteins used for the pulldown assay. D, duplicate experiments validating knockdown of hNOA1 expression by hNOA1 siRNA. hNOA1 is depleted ∼90% (lanes 2 and 4) compared with controls (lanes 1 and 3). The amounts of other mitochondrial proteins, such as mtHSP70, DAP3 and cytochrome c are similar in control and hNOA1 siRNA-treated samples. HeLa cells (160,000) from control or hNOA1 siRNA-treated samples were lysed directly in 2× sample buffer, followed by SDS-PAGE, and immunoblotting for mitochondrial markers. E, knockdown of hNOA1 leads to decreased O₂ consumption in a manner dependent on Complex I, but not Complex II-linked substrates. The rate of O₂ consumption is similar between control and hNOA1 siRNA-treated samples when the cells are either intact or digitonin-permeabilized and supplemented with succinate (plus rotenone). In contrast, the O₂ consumption is reduced ∼20% in hNOA1 depleted cells versus controls when cells are supplemented with the Complex I-linked substrates, glutamate and malate. 1.0 × 10⁷ intact or digitonin-permeabilized HeLa cells transfected with control or hNOA1 siRNA were suspended in 0.6 ml of KCl medium in the presence of 5 mm glutamate and malate or 5 mm succinate and 2 μm rotenone. The maximal O₂ consumption rates (state 3_u) were measured with a Hansatech Oxytherm electrode by titration with the uncoupler 80 nm carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

hNOA1 directly interacts with DAP3 and regulates γ-interferon induced apoptosis. A, hNOA1 coimmunoprecipitates with endogenous DAP3. Immunoprecipitates obtained from equal aliquots of HEK293 cell lysates with anti-hNOA1 and preimmune (Pre) IgG were separated by SDS-PAGE and immunoblotted with antibodies against hNOA1 and DAP3. B, GST-hNOA1 but not GST alone specifically interacts with endogenous DAP3 from HEK293 cells in GST-pulldown assays. Pulldown assays were carried out as in Fig. 5C. C, in vitro translated, [³⁵S]Met-labeled DAP3 binds to GST-hNOA1, but not GST alone. GST-hNOA1 and GST alone (∼2 μg each) immobilized on glutathione beads were incubated with in vitro translated DAP3. Bound proteins were separated by SDS-PAGE and detected by autoradiography. D, GST-hNOA1 but not GST alone, interacts with purified His₆-DAP3. GST-hNOA1 and GST alone (∼2 μg each) immobilized on glutathione beads were incubated with ∼200 ng of recombinant His₆-DAP3 purified from E. coli. Bound proteins were separated by SDS-PAGE, followed by immunoblotting with anti-DAP3 IgG. E, depletion of hNOA1 or DAP3 renders cells more resistant to γ-interferon-induced apoptosis. HeLa cells were plated and transfected with control, hNOA1, or DAP3 siRNA and placed in fresh DMEM containing 1000 units/ml γ-interferon. 2.5 days later, cells were trypsinized and collected in 1 ml of fresh DMEM and counted using a Vi-Cell cell counter (Beckman Coulter).

hNOA1 knockdown protects cells from staurosporine-induced apoptosis. HeLa cells transfected with control siRNA (A and B) or hNOA1 siRNA (C and D) for 3 days were treated with 2 μm staurosporine for 5 h after which cells were fixed in 2% PFA, followed by TUNEL staining (red nuclear staining in A and C) to detect apoptotic cells and 4′,6-diamidino-2-phenylindole staining (DAPI)(B and D). Fewer cells show TUNEL staining in hNOA1-depleted cells then in controls (compare A and C). E, quantification reveals that cells transfected with hNOA1 siRNA (hNOA1 RNAi) show ∼30% less TUNEL staining than those transfected with control siRNA. For either control or hNOA1 siRNA transfected samples total cells (595 for ctrl RNAi; 493 for hNOA1 RNAi), and apoptotic cells were counted in eight randomly selected fields based on 4′,6-diamidino-2-phenylindole and TUNEL staining, respectively. F-K, cytochrome c release is not observed in HeLa cells transfected with either control or hNOA1 siRNA in the absence of staurosporine. L-Q, in control siRNA-transfected samples treated with staurosporine more cells in which cytochrome c has been released (asterisk in L) are observed than in hNOA1 siRNA-transfected samples (O). HeLa cells transfected with control (L-N) or hNOA1 (O and P) siRNA for 3 days were treated or not with staurosporine as above and processed for double immunofluorescence with anti-Cyt C (L and O) and anti-hNOA1 (M and P) IgG. R, quantification reveals that only ∼7% of the cells transfected with hNOA1 siRNA (hNOA1 RNAi) showed cytochrome c release, whereas ∼37% of the cells transfected with control siRNA (ctrl RNAi) showed cytochrome c release. For either control or hNOA1 siRNA-transfected samples, eight fields were randomly selected, and the total number of cells (420 for ctrl RNAi; 316 for hNOA1 RNAi), and the percentage of the total with released cytochrome c was counted.